CX3CR1 MEDIATES THE DEVELOPMENT OF MONOCYTE-DERIVED DENDRITIC CELLS DURING HEPATIC INFLAMMATION. Supplementary material Supplementary Figure 1: Liver CD45+ myeloid cells were pre-gated for Ly6G negative cells for excluding granulocytes and HDCs subsequently analyzed among the cells that were CD11c+ and had high expression of MHCII. Supplementary Table 1 low/- high + Changes in gene expression between CX3CR1 and CX3CR1 CD11b myeloid hepatic dendritic cells (HDCs) from CCl4-treated mice high Genes up-regulated in CX3CR1 HDCs Gene Fold changes P value Full name App 4,01702 5,89E-05 amyloid beta (A4) precursor protein C1qa 9,75881 1,69E-22 complement component 1, q subcomponent, alpha polypeptide C1qb 9,19882 3,62E-20 complement component 1, q subcomponent, beta polypeptide Ccl12 2,51899 0,011769 chemokine (C-C motif) ligand 12 Ccl2 6,53486 6,37E-11 chemokine (C-C motif) ligand 2 Ccl3 4,99649 5,84E-07 chemokine (C-C motif) ligand 3 Ccl4 4,42552 9,62E-06 chemokine (C-C motif) ligand 4 Ccl6 3,9311 8,46E-05 chemokine (C-C motif) ligand 6 Ccl7 2,60184 0,009272 chemokine (C-C motif) ligand 7 Ccl9 4,17294 3,01E-05 chemokine (C-C motif) ligand 9 Ccr2 3,35195 0,000802 chemokine (C-C motif) receptor 2 Ccr5 3,23358 0,001222 chemokine (C-C motif) receptor 5 Cd14 6,13325 8,61E-10 CD14 antigen Cd36 2,94367 0,003243 CD36 antigen Cd44 4,89958 9,60E-07 CD44 antigen Cd81 6,49623 8,24E-11 CD81 antigen Cd9 3,06253 0,002195 CD9 antigen Cdkn1a 4,65279 3,27E-06 cyclin-dependent kinase inhibitor 1A (P21) Cebpb 6,6083 3,89E-11 CCAAT/enhancer binding protein (C/EBP), beta Cfb 2,10895 0,034949 complement factor B Cmklr1 6,72268 1,78E-11 chemokine-like receptor 1 Csf1r 6,36466 1,96E-10 colony stimulating factor 1 receptor Csf3r 3,84985 0,000118 colony stimulating factor 3 receptor (granulocyte) Ctsc 5,89204 3,81E-09 cathepsin C Ctss 3,2198 0,001283 cathepsin S Cx3cr1 13,1318 2,16E-39 chemokine (C-X3-C) receptor 1 Cybb 4,92213 8,56E-07 cytochrome b-245, beta polypeptide Ddx58 2,66239 0,007759 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 Emr1 9,69028 3,32E-22 EGF-like module containing, mucin-like, hormone receptor-like sequence 1 Fcer1g 4,20145 2,65E-05 Fc receptor, IgE, high affinity I, gamma polypeptide Fcgr1 9,18398 4,15E-20 Fc receptor, IgG, high affinity I Fcgr2b 4,65036 3,31E-06 Fc receptor, IgG, low affinity IIb Fcgr4 7,62716 2,40E-14 Fc receptor, IgG, low affinity IV Fn1 5,74254 9,33E-09 fibronectin 1 Ifi204 5,51675 3,45E-08 interferon activated gene 204 Ifnar2 4,27785 1,89E-05 interferon (alpha and beta) receptor 2 Ifngr2 2,48297 0,013029 interferon gamma receptor 2 Ikbke 5,38026 7,44E-08 inhibitor of kappaB kinase epsilon Il10rb 4,22073 2,44E-05 interleukin 10 receptor, beta Il1b 2,70627 0,006804 interleukin 1 beta Il1rn 3,33231 0,000861 interleukin 1 receptor antagonist Irf7 3,6732 0,00024 interferon regulatory factor 7 Itga6 4,92143 8,59E-07 integrin alpha 6 Itgal 5,4946 3,92E-08 integrin alpha L Lair1 5,00721 5,52E-07 leukocyte-associated Ig-like receptor 1 Lilra5 3,17543 0,001496 leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 5 Litaf 2,82839 0,004678 LPS-induced TN factor Ly86 2,79276 0,005226 lymphocyte antigen 86 Maf 4,71703 2,39E-06 avian musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene homolog Msr1 8,39109 4,82E-17 macrophage scavenger receptor 1 Ncf4 3,33874 0,000842 neutrophil cytosolic factor 4 Nfil3 3,31981 0,000901 nuclear factor, interleukin 3, regulated Nfkbiz 2,76339 0,00572 nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, zeta Npc1 2,9948 0,002746 Niemann Pick type C1 Pdgfb 3,85957 0,000114 platelet derived growth factor, B polypeptide Plau 6,42638 1,31E-10 plasminogen activator, urokinase Plaur 4,15787 3,21E-05 plasminogen activator, urokinase receptor Pparg 4,70638 2,52E-06 peroxisome proliferator activated receptor gamma Prdm1 2,975 0,00293 PR domain containing 1, with ZNF domain Ptafr 2,89613 0,003778 platelet-activating factor receptor Ski 2,58243 0,009811 ski sarcoma viral oncogene homolog (avian) Smad3 2,70074 0,006918 MAD homolog 3 (Drosophila) Stat2 2,51455 0,011919 signal transducer and activator of transcription 2 Tgfb1 3,16093 0,001573 transforming growth factor, beta 1 Tgfbi 5,15178 2,58E-07 transforming growth factor, beta induced Tgfbr1 5,45649 4,86E-08 transforming growth factor, beta receptor I Tlr1 4,84139 1,29E-06 toll-like receptor 1 Tlr2 2,69617 0,007014 toll-like receptor 2 Tlr4 3,61259 0,000303 toll-like receptor 4 Tlr8 6,27586 3,48E-10 toll-like receptor 8 Tnfrsf11a 3,04365 0,002337 tumor necrosis factor receptor superfamily, member 11a Tnfrsf14 3,13939 0,001693 tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator) Tnfrsf1b 4,96437 6,89E-07 tumor necrosis factor receptor superfamily, member 1b Tnfsf12 2,79525 0,005186 tumor necrosis factor (ligand) superfamily, member 12 Trem2 5,50484 3,70E-08 triggering receptor expressed on myeloid cells 2 Tyrobp 2,66403 0,007721 TYRO protein tyrosine kinase binding protein high Genes down-regulated in CX3CR1 HDCs Gene Fold changes P value Full name Ahr -2,611174 0,009023 aryl-hydrocarbon receptor Btla -6,957308 3,47E-12 B and T lymphocyte associated Ccl22 -4,362738 1,28E-05 chemokine (C-C motif) ligand 22 Ccr7 -7,241423 4,44E-13 chemokine (C-C motif) receptor 7 Cd244 -2,605379 0,009177 CD244 natural killer cell receptor 2B4 Cd24a -5,198278 2,01E-07 CD24a antigen Cd34 -5,099496 3,41E-07 CD34 antigen Cd7 -5,286396 1,25E-07 CD7 antigen Cd82 -2,956234 0,003114 CD82 antigen Cd83 -3,163861 0,001557 CD83 antigen Ciita -5,820575 5,86E-09 class II transactivator Dpp4 -5,877618 4,16E-09 dipeptidylpeptidase 4 Fyn -3,696658 0,000218 Fyn proto-oncogene Gpr183 -2,784596 0,005359 G protein-coupled receptor 183 H2-Aa -2,968497 0,002993 histocompatibility 2, class II antigen A, alpha H2-DMb2 -3,67274 0,00024 histocompatibility 2, class II, locus Mb2 H2-Ob -5,529531 3,21E-08 histocompatibility 2, O region beta locus Icosl -4,664422 3,09E-06 icos ligand Ifitm1 -8,456888 2,75E-17 interferon induced transmembrane protein 1 Il1r2 -6,064631 1,32E-09 interleukin 1 receptor, type II Il7r -2,692397 0,007094 interleukin 7 receptor Itgax -2,68947 0,007157 integrin alpha X Jak2 -6,927284 4,29E-12 Janus kinase 2 Kit -4,144838 3,40E-05 kit oncogene Ltb -2,913954 0,003569 lymphotoxin B Ltb4r1 -2,481954 0,013066 leukotriene B4 receptor 1 Relb -5,286109 1,25E-07 avian reticuloendotheliosis viral (v-rel) oncogene related B Runx3 -4,727032 2,28E-06 runt related transcription factor 3 S100a8 -4,289814 1,79E-05 S100 calcium binding protein A8 (calgranulin A) Sell -4,53454 5,77E-06 selectin, lymphocyte Sigirr -3,943093 8,04E-05 single immunoglobulin and toll-interleukin 1 receptor (TIR) domain Slamf7 -3,795066 0,000148 SLAM family member 7 Stat5a -3,201358 0,001368 signal transducer and activator of transcription 5A Tagap -3,1848 0,001449 T cell activation Rho GTPase activating protein Tbx21 -2,340494 0,019258 T-box 21 Tcf7 -2,9563 0,003114 transcription factor 7, T cell specific Tlr3 -4,154069 3,27E-05 toll-like receptor 3 Traf1 -6,474935 9,49E-11 TNF receptor-associated factor 1 Xcr1 4,51824 6,24E-06 chemokine (C motif) receptor 1 Zeb1 4,141243 3,45E-05 zinc finger E-box binding homeobox 1 low/- high + CX3CR1 and CX3CR1 /CD11b myeloid HDCs were obtained from the livers of CCl4-treated mice and were cell sorted using a FACS Aria cytometer. Gene expression was analysed using Nanostring gene array. Supplementary Figure 2: The lack of CX3CR1 affects the pool of monocyte-derived dendritic cells (moDCs). Flow cytometry plot showing the cluster of moDCs identified as co-expressing CD11c, MHCII, gfp/+ gfp/gfp CX3CR1, CD11b and CD88 in the livers of CX3CR1 and CX3CR1 mice 36 hours after receiving an acute dose of CCl4. Supplementary Figure 3: The lack of CX3CR1 did not interfere with the liver recruitment of + CX3CR1 monocytes/macrophages. + high + The distribution of F4-80 /CD11b /CX3CR1 monocytes/macrophages was evaluated by flow gfp/+ gfp/gfp cytometry in the livers of CX3CR1 and CX3CR1 mice 36 hours after receiving an acute dose of CCl4. Supplementary Figure 4: The lack of CX3CR1 affects in vitro differentiation of monocyte-derived dendritic cells (moDCs). MoDCs were allowed to differentiate by 7 days culture of bone marrow myeloid cells from either gfp/+ gfp/gfp CX3CR1 and CX3CR1 mice in RPMI-1640 medium supplemented with 10% fetal bovine - serum. (Panel A) Effect of CX3CR1 deficiency on the maturation of Ly6G + + + high /CD11b /CD88 /CD11c /MHCII moDCs. (Panel B) Effect of CX3CR1 deficiency on the expression of MHCII by CD11b+/CD88+/CD11c+ pre-dendritic cells. Supplementary Figure 5: Effect of CX3CR1 antagonist CX3-AT in naïve mice. Wild-type mice were injected IP with a single dose of CX3-AT (150 µg in saline) or saline (Cont) and the effects on liver integrity and hepatic leukocyte distribution were evaluated after 12 hours. (Panel A) Liver morphology (Hematoxylin/eosin staining of formalin-fixed liver sections) and circulating levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).